This proposal will structurally characterize the conformational transitions of five homologous FKBP domains by a combination of NMR relaxation, NOE analysis, quantitative amide hydrogen exchange analysis, X-ray crystallography, and molecular dynamics simulations. As these five FKBP proteins participate in regulating distinctly different sets of signaling pathways, development of pharmacological selectivity among these proteins is of significant interest. However, the marked similarity among their crystal structures has appreciably impeded this process. We have observed a substantial degree of diversity in the conformational dynamics of these five domains. We are identifying mutations which modulate these transitions that can then assist both in structurally characterizing these transitions and in the stepwise optimization of drug leads. This systematic mutational modulation of the intrinsic conformational transitions of the FKBP domains will facilitate the analysis of the potential role of these transitions in coupling to the larger scale signaling processes of the biological complexes in which these proteins function as will be examined regarding their roles in regulating cardiac Ca2+ and Na+ membrane channels and in the transcription factor complexes of the glucocorticoid and PPAR? receptors. Specific Aim 1: The structure of transient conformations sampled by the FKBP domains of FKBP51, FKBP52 and FKBP38 and the interactions between these transitions will be characterized by a combination of NMR, crystallographic and molecular simulation studies. Variants of FKBP51/52, designed to modulate these transitions, will be used to test a structural model for regulatory selectivity in steroid receptor interactions. Similar structural studies will be applied to both human FKBP38 isoforms. Models of transient conformations and known catalytic site-binding molecular scaffolds will be used for in silico ligand design. Specific Aim 2: This set of experimental and modeling approaches will be applied to FKBP12 and FKBP12.6 to understand the structural basis of the energetic coupling between two distantly separated conformational transitions centered on either side of the catalytic cleft. Structural insight into the comparative inefficiency of this long range coupling in FKBP12.6 will be obtained. Specific Aim 3: Murine embryonic fibroblast knockout cell lines will be transfected with genes for the FKBP51/FKBP52 variants. Glucocorticoid and PPAR? receptors will be tested for competitive FKBP binding, cytoplasmic vs. nuclear localization, transcription activity, and p38 kinase-mediated phosphorylation. Cardiomyocyte Ca2+ spark frequency and voltage-gated sodium current will be measured in FKBP12.6-KO and FKBP12cKO mouse cells, respectively, with FKBP12/12.6 variant proteins being introduced by dialysis.